Pathogenicity Of Pine Wood Nematode Research Articles

Bacterial Diversity and Pathogenicity of Pine Wood Nematode (Bursaphelenchus xylophilus) from Two Diff erent Sources

Pine wilt disease is a devastating disease of pine trees caused by a pine wood nematode (PWN), Bursaphelenchus xylophilus (Steiner & Buhrer) Nickle. We studied the bacteria carried by B. xylophilus from Fushan Mountain and Qingdao University Campus (F and Q samples) and the pathogenicity of the two PWN samples to explore the relationship between PWN companion bacteria and its pathogenicity. Based on metagenomic analysis, the distribution of the bacteria from the two samples was signifi cantly different. At the phylum level, Proteobacteria is the largest phylum in the two samples, 85 and 65% in F and Q samples, respectively. At the family level, Enterobacteriaceae and Rhizobiaceae were dominant in F sample with the total relative abundance of 55% while the two bacterial families only accounted for 15% in Q sample. Moreover, Burkholderiaceae was only detected in F sample. PICRUSt analysis based on KEGG database showed that the bacteria carried by B. xylophilus from F sample had stronger metabolic abilities. In addition, BugBase analysis predicted that the bacteria from F sample had stronger effect on pathogenicity and antioxidant stress. Furthermore, we tested the feeding ability and pathogenicity of F and Q samples, and found that the B. xylophilus of F sample had stronger feeding ability and pathogenicity, which was consistent with the results of PICRUSt analysis based on KEGG database and BugBase analysis. Our research indicated that the pathogenicity of B. xylophilus could be closely related to the vitality of its associated bacteria, and Enterobacteriaceae, Rhizobiaceae and Burkholderiaceae belonging to Proteobacteria phylum were important pathogenicity-related bacterial families, which is valuable for the study on the pathogenesis of B. xylophilus.

Multi-Omics of Pine Wood Nematode Pathogenicity Associated With Culturable Associated Microbiota Through an Artificial Assembly Approach.

Pinewood nematode (PWN), the causal agent of pine wilt disease (PWD), causes massive global losses of Pinus species each year. Bacteria and fungi existing in symbiosis with PWN are closely linked with the pathogenesis of PWD, but the relationship between PWN pathogenicity and the associated microbiota is still ambiguous. This study explored the relationship between microbes and the pathogenicity of PWN by establishing a PWN-associated microbe library, and used this library to generate five artificial PWN–microbe symbiont (APMS) assemblies with gnotobiotic PWNs. The fungal and bacterial communities of different APMSs (the microbiome) were explored by next-generation sequencing. Furthermore, different APMSs were used to inoculate the same Masson pine (Pinus massoniana) cultivar, and multi-omics (metabolome, phenomics, and transcriptome) data were obtained to represent the pathogenicity of different APMSs at 14 days post-inoculation (dpi). Significant positive correlations were observed between microbiome and transcriptome or metabolome data, but microbiome data were negatively correlated with the reactive oxygen species (ROS) level in the host. Five response genes, four fungal genera, four bacterial genera, and nineteen induced metabolites were positively correlated with the ROS level, while seven induced metabolites were negatively correlated. To further explore the function of PWN-associated microbes, single genera of functional microbes (Mb1–Mb8) were reloaded onto gnotobiotic PWNs and used to inoculate pine tree seedlings. Three of the genera (Cladophialophora, Ochroconis, and Flavobacterium) decreased the ROS level of the host pine trees, while only one genus (Penicillium) significantly increased the ROS level of the host pine tree seedlings. These results demonstrate a clear relationship between associated microbes and the pathogenicity of PWN, and expand the knowledge on the interaction between PWD-induced forest decline and the PWN-associated microbiome.

Cathepsin L-like Cysteine Proteinase Genes Are Associated with the Development and Pathogenicity of Pine Wood Nematode, Bursaphelenchus xylophilus.

The pine wood nematode (PWN), Bursaphelenchus xylophilus, is the pathogen of pine wilt disease (PWD), resulting in huge losses in pine forests. However, its pathogenic mechanism remains unclear. The cathepsin L-like cysteine proteinase (CPL) genes are multifunctional genes related to the parasitic abilities of plant-parasitic nematodes, but their functions in PWN remain unclear. We cloned three cpl genes of PWN (Bx-cpls) by rapid amplification of cDNA ends (RACE) and analyzed their characteristics using bioinformatic methods. The tissue specificity of cpl gene of PWN (Bx-cpl) was studied using in situ mRNA hybridization (ISH). The functions of Bx-cpls in development and pathogenicity were investigated using real-time quantitative PCR (qPCR) and RNA interference (RNAi). The results showed that the full-length cDNAs of Bx-cpl-1, Bx-cpl-2, and Bx-cpl-3 were 1163 bp, 1305 bp, and 1302 bp, respectively. Bx-cpls could accumulate specifically in the egg, intestine, and genital system of PWN. During different developmental stages of PWN, the expression of Bx-cpls in the egg stage was highest. After infection, the expression levels of Bx-cpls increased and reached their highest at the initial stage of PWD, then declined gradually. The silencing of Bx-cpl could reduce the feeding, reproduction, and pathogenicity of PWN. These results revealed that Bx-cpls play multiple roles in the development and pathogenic processes of PWN.

The alcohol dehydrogenase with a broad range of substrate specificity regulates vitality and reproduction of the plant-parasitic nematode Bursaphelenchus xylophilus.

Pine wilt disease, which is caused by the pine wood nematode (PWN), Bursaphelenchus xylophilus, has caused huge damage to pine forests around the world. In this study, we analysed the PWN transcriptome to investigate the expression of genes related to the associated bacterial species Pseudomonas fluorescens and found that the gene adh-1 encoding alcohol dehydrogenase (ADH) was upregulated. The open reading frame of adh-1, which encoded a protein of 352 amino acid residues, was cloned from B. xylophilus. Recombinant ADH with a relative molecular weight of 39 kDa, was present mainly in inclusion bodies and was overexpressed in Escherichia coli BL21 (DE3) and purified after refolding. The biochemical assay revealed that recombinant ADH could catalyse the dehydrogen reaction of eight tested alcohols including ethanol in the presence of NAD+. Quantitative real-time RT-PCR analysis indicated that ethanol upregulated adh-1 expression in PWN. Results of RNA interference and inhibition of ADH treatment indicated that downregulating expression of adh-1 or inhibition of ADH could reduce ethanol tolerance and the vitality and reproduction ability of B. xylophilus, suggesting that adh-1 is involved in pathogenicity of PWN.

Scientific Opinion on comments provided by Portugal on the phytosanitary risk associated with Pinus pinea for the spread of pine wood nematode

Following a request from European Commission, the PLH Panel examined the comments of the Portuguese plant health authorities on a previous EFSA opinion with regard to pine wood nematode (PWN) and Pinus pinea and a report on PWN surveys in Portugal. Regarding the comment, based on behavioural responses of PWN to CO2 and to ß-myrcene, that Monochamus galloprovincialis can feed on P. pinea without implying PWN infestation, the Panel noted the uncertainties on how chemical attraction influences the exit of PWN from the vector. The Panel agreed that P. pinea is not a preferred breeding host of M. galloprovincialis, but it noted the evidence from Italy of breeding of M. galloprovincialis in fallen woods of P. pinea, suggesting P. pinea to be an occasional host. The Panel agreed that, although pathogenicity of PWN has been recorded on P. pinea in inoculation experiments on small plants, this does not necessarily relate to pathogenicity on larger trees in the field. The additional information on Portuguese PWN surveys is not sufficient to conclude that P. pinea is resistant to PWN. The Panel also noted that asymptomatic infestations by PWN are well known in other areas, and pine species considered tolerant to PWN could still maintain it at low levels in restricted parts of a tree. The Panel concluded that there is high uncertainty concerning the classification of the risk of spread of PWN with plants and wood of P. pinea, owing to the scarcity of information on the interaction of M. galloprovincialis, PWN and P. pinea, as well as on the field resistance of P. pinea to PWN. Owing to high uncertainty related to the host potential of P. pinea, more studies on the transmission of PWN at feeding wounds and on its survival in trees and wood of P. pinea are needed.

The decrease of pathogenicity of pine wood nematode, Bursaphelenchus lignicolus, induced by the extended ubculturing on the fungal mat of Botrytis cinerea

The decrease of pathogenicity of pine wood nematode, Bursaphelenchus lignicolus, induced by the extended ubculturing on the fungal mat of Botrytis cinerea